Monoubiquitination is a regulatory signal, like phosphorylation, that can alter the activity, location or structure of a protein. One function of monoubiquitin is to serve as a sorting signal in the endocytic pathway to promote the downregulation of activated signaling receptors and other plasma membrane proteins. Ubiquitin triggers the entry of endocytic cargo into primary endocytic vesicles budding from the plasma membrane. Ubiquitin is also a signal for the entry of receptors into vesicles that bud into the lumen of a late endosomal compartment, an event that is required for the delivery and degradation of receptors into the interior of the lysosome. Monoubiquitin plays multiple roles in regulating endocytic traffic. In addition to serving as a cargo sorting signal, monoubiquitin regulates the activity of the trans-acting endocytic machinery. Ubiquitin controls the cell surface activity of proteins involved in growth and differentiation, cell adhesion, hypertension, cardiac disease and neurotransmission. Ubiquitin-dependent endocytosis is also a mechanism that has been co-opted by viruses to downregulate host cell immune response proteins as part of the viral strategy to avoid immune surveillance. Furthermore, enveloped viruses, such as HIV and Ebola, appear to use the host cell ubiquitin-dependent endocytic machinery to assemble and bud from the plasma membrane. The mechanisms by which monoubiquitin bestows its regulatory activities have not been defined. In particular, ubiquitin-binding proteins that recognize and transmit the regulatory information conferred by monoubiquitination remain largely uncharacterized. To define the roles of ubiquitin signals in endocytosis and to provide paradigms for how monoubiquitin signals function in general, we will use biochemistry and genetics in Saccharomyces cerevisiae to characterize ubiquitin-binding endocytic proteins. We will 1) test models for the function of ubiquitin-binding proteins in the internalization and endosomal sorting machinery, 2) define the relationship between monoubiquitin-binding and intramolecular monoubiquitination for the CUEUb and UIM ubiquitin-binding domains; 3) identify ubiquitinated interacting partners of monoubiquitin-binding domains and 4) identify and characterize new ubiquitin-binding proteins that interact with the ubiquitin internalization signal.